Sphingosine kinase Type 1 inhibitors and uses thereof

ABSTRACT

Provided are inhibitors of sphingosine kinase Type I that are useful in the treatment of neuropathic pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/813,961 filed Mar. 10, 2020, which is a continuation of U.S.application Ser. No. 16/572,699 filed Sep. 17, 2019, which is acontinuation of U.S. application Ser. No. 16/199,337 filed Nov. 26, 2018(now U.S. patent Ser. No. 10/456,370), which is a continuation of U.S.application Ser. No. 15/954,131 filed Apr. 16, 2018, (now U.S. Pat. No.10,166,204), which is a continuation of U.S. application Ser. No.15/181,826 filed Jun. 14, 2016 (now U.S. Pat. No. 9,974,758), which is acontinuation of U.S. application Ser. No. 13/649,221 filed Oct. 11, 2012(now U.S. Pat. No. 9,388,121), which is a divisional of U.S. applicationSer. No. 12/584,131 filed Aug. 31, 2009 (now U.S. Pat. No. 8,314,151),which is a continuation-in-part of U.S. application Ser. No. 12/387,228filed Apr. 29, 2009 (now U.S. Pat. No. 8,372,888), which claims thebenefit of U.S. Provisional Application No. 61/048,638 filed Apr. 29,2008, the contents of all of which are hereby incorporated by referencein their entireties.

GOVERNMENT SUPPORT STATEMENT

This invention was made with government support under Grant R01 CA61774awarded by the National Cancer Institute (NCI). The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 13, 2020, isnamed ENZ-91-CIP-D1-CON-CON-CON-CON-CON-CON-Application-SL.txt and is921 bytes in size.

FIELD OF THE INVENTION

The invention relates to the field of sphingosine kinase Type 1 (SphK1)inhibitors.

BACKGROUND OF THE INVENTION

Sphingosine-1-phosphate (S1P), a potent lipid mediator produced fromsphingosine by sphingosine kinases (SphKs), regulates many processesimportant for cancer progression, including cell growth and survival(Spiegel et al., Nature Rev Mol Cell Biol. 4:397-407, 2003). In contrastto S1P, its precursors, sphingosine and ceramide, are associated withgrowth arrest and induction of apoptosis (Ogretman & Hannun, Nature RevCancer 4:604-616, 2004). Thus, the balance between theseinterconvertible sphingolipid metabolites has been viewed as a cellularrheostat determining cell fate (Cuvillier et al., Nature 381:800-803,1996). Numerous studies have shown that perturbations in theS1P/ceramide rheostat are involved in the regulation of resistance tochemotherapy and radiation therapy of neoplastic cells, including thoseof hematopoietic origin (Ogretman et al., supra.; Hait et al., BiochimBiophys Acta 1758:2016-2026. 2006; and Milstien & Spiegel, Cancer Cell9:148-150, 2006).

Two Sphk isoenzymes, SphK1 and SphK2, have been described which, whilesharing many features (Kohama et al., J. Biol Chem 273:23722-23728,1998; and Liu et al., J. Biol Chem 275:19513-19520, 2000) exhibitdistinct functions. SphK1 promotes cell growth and survival (Olivera etal., J Cell Biol 147:545-558, 1999; Xia et al., J. Biol Chem277:7996-8003, 2002; Bonhoure et al., Leukemia 20:95-102, 2006; andSukocheva et al., J Cell Biol 173:301-310, 2006), whereas SphK2, whenoverexpressed, has opposite effects (Maceyka et al., J Biol Chem280:37118-37129, 2005; and Okada et al., J Biol Chem 280:36318-36325,2005). SphK1 is a key enzyme that regulates the S1P/ceramide rheostat(Maceyka et al., supra.; Berdyshev et al., Cell Signal 18:1779-1792,2006; and Taha et al., FASEB J 20:482-484, 2006). Indeed, S1P and SphK1have long been implicated in resistance of both primary leukemic cellsand leukemia cell lines to apoptosis induced by commonly used cytotoxicagents (Cuvillier et al., Nature, 2004 supra.; Cuvillier et al., J. BiolChem 273:2910-2916, 1998; Cuvillier et al., Blood 98:2828-2836, 2001;and Jendiroba et al., Leuk Res 26:301-310, 2002). Non-isozyme specificinhibitors of SphKs, such as L-threo-dihydrosphingosine (safingol) andN,N-dimethylsphingosine (DMS), are cytotoxic to leukemia cells (Jarviset al., Mol Pharmacol 54:844-856, 1998; and Jendiroba et al., 2002,supra.). Interestingly, multi-drug resistant HL-60 myelogenous leukemiacells were more sensitive to DMS than the parental cells (Jendiroba etal., 2002, supra.). Moreover, SphK1 activity was lower in HL-60 cellssensitive to doxorubicin or etoposide than in MDRI- or MRP1-positiveHL-60 cells. Enforced expression of SphK1 in sensitive HL-60 cellsblocked apoptosis whereas downregulation of Sphk1 overcamechemoresistance by inducing mitochondria-dependent apoptosis (Bonhoureet al., 2006, supra.). These observations take on added significance inlight of evidence that MDR expression is a strong prognostic indicatorin acute myelogenous leukemia (AML) (Filipits et al., Leukemia 14:68-76,2000) and that the MDR phenotype, which commonly arises followingtreatment of AML with anthracyclines or plant-based alkaloids, isthought to represent an obstacle to successful chemotherapy. Inaddition, resistance of K562 human chronic myeloid leukemia cells tolmatinib, an inhibitor of Bcr-Abl tyrosine kinase, correlated withexpression of SphK1 and generation of S1P, whereas downregulation ofSphK1 increased sensitivity to lmatinib-induced apoptosis in resistantcells (Baran et al., J Biol Chem 282:10922-10934, 2007). Thus, thedevelopment of effective and specific inhibitors of SphK1 might proveuseful not only in diminishing levels of pro-survival S1P, but also inpotentiating ceramide generation, a process that mediates, at least inpart, the pro-apoptotic actions of certain cytotoxic agents (Maggio etal., Cancer Res 64:2590-2600, 2004; Rahmani et al., Cancer Res65:2422-2432, 2005; and Rosato et al., Mol Pharmacol 69:216-225, 2006).

Sphingosine kinase inhibitors have been described (Kim et al., Bioorg &Med Chem 13:3475-3485, 2005; Kono et al., J. Antibiotics 53:459-466,2000; Kono et al., J. Antibiotics 53:753-758, 2000; Marsolais & Rosen,Nature Reviews/Drug Discovery 8:297-307, 2009; and US 2008/0167352 A1(Smith et al., published Jul. 10, 2008). None of these publicationsdescribe, however, the novel sphingosine kinase Type 1 inhibitorsherein. Halide modified analogs of sphingosine derivatives have alsobeen described (Qu et al., Bioorg & Med Chem Letters 19:3382-3385(2009).

In U.S. patent application Ser. No. 12/387,228 (filed Apr. 29, 2009),there is described a potent, water-soluble inhibitor of SphK1 (SK1-I)that triggers multiple perturbations in activation of various signalingand survival-related proteins. SK1-I markedly induced apoptosis in humanleukemic cell lines as well as blasts obtained from patients with AMLand inhibited growth of AML xenograft tumors. SK1-1 serves as model forother related compounds which are described further below.

Glioblastoma multiforme (GBM) is the most prevalent and lethal type ofprimary central nervous system tumors with a median survival of 10-12months, even after aggressive surgery, radiation and advancedchemotherapy (Maher et al., Genes Dev 15:1311-1333, 2001). Poorprognosis of patients with GBM has recently been correlated withelevated expression of sphingosine kinase type 1 (SphK1) (Van Brocklynet al., J Neuropathol Exp Neurol 64:695-705, 2005; Li et al., ClinCancer Res 14:6996-7003, 2008), one of the SphK isoenzymes thatgenerates the pleiotropic lipid mediator, sphingosine-1-phosphate (S1P).S1P has been implicated in the etiology of GBM due to its involvement invarious cell processes particularly important for cancer progression,including growth, survival, migration, invasion, tumor growth,angiogenesis, and metastasis (Van Brocklyn et al., Cancer Lett181:195-204, 2002; Lepley et al., Cancer Res. 65:3788-3795, 2002;Radeff-Huang et al., J Biol Chem 282:863-870, 2007; and Young et al.,Exp Cell Res 313:1615-1627, 2007). The biological effects of thisserum-borne lipid are mainly mediated by a family of five specific Gprotein-coupled receptors, designated S1P₁₋₅. (Murph and Mills, ExpertRev Mod Med 9:1-18, 2007). Of those, S1P₁₋₃, are expressed in themajority of human glioblastoma cell lines and are involved inS1P-mediated proliferation (Van Brocklyn et al., Cancer Lett181:195-204, 2002, supra). Although S1P has no effect on matrixmetalloproteinase secretion, it enhances glioblastoma cell adhesion andalso stimulates their motility and invasiveness (Van Brocklyn et al.,Cancer Lett 199:53-60, 2003). Because S1P is present at high levels inbrain tissue, it is possible that autocrine or paracrine signaling byS1P through its receptors enhances both glioma cell proliferation andinvasiveness (Anelli et al., J Biol Chem 283:3365-3375, 2008).

To explore the therapeutic implications of targeting SphK1 for treatmentof GBM, the effects of a newly developed isozyme-specific inhibitor ofSphK1, SK1-1 (Paugh et al., Blood 112:1382-1391, 2008), was examined andfound that it inhibits growth of GBM in vitro and in vivo. Thesespecific SphK1 inhibitors are useful for treatment, either alone or incombination with advanced chemotherapeutic agents.

SUMMARY OF THE INVENTION

This invention provides a composition of matter having the structure:

wherein R₁ is H or comprises N, S, a phosphate group or a phosphonategroup, and any combination thereof; wherein R₂ is H, or comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbonchain, a branched carbon chain, a straight carbon chain comprising oneor more heteroatoms, a branched carbon chain comprising one or moreheteroatoms, a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₄is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₅ comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing.

This invention also provides a composition of matter having thestructure:

wherein R₁ is H or comprises OH, N, S, a phosphate group or aphosphonate group, and any combination thereof; wherein R₂ comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing,wherein said R₂ is substituted with one or more halides; wherein R₃ is Hor comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₄is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₅ comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing.

This invention additionally provides a composition of matter having thestructure:

wherein R₁ is H or comprises OH, N, S, a phosphate group or aphosphonate group, and any combination thereof; wherein R₂ is H, orcomprises a straight carbon chain, a branched carbon chain, a straightcarbon chain comprising one or more heteroatoms, a branched carbon chaincomprising one or more heteroatoms, a cyclic ring, a heterocyclic ring,an aromatic ring or a hetero-aromatic ring, and any combination of theforegoing; wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straightcarbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₆, R₇ and R₈ independently comprise H, a straight carbon chain,a branched carbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₄is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₅comprises a straight carbon chain, a branched carbon chain, a straightcarbon chain comprising one or more heteroatoms, a branched carbon chaincomprising one or more heteroatoms, a cyclic ring, a heterocyclic ring,an aromatic ring or a hetero-aromatic ring, and any combination of theforegoing; and wherein R₃ and R₄ each comprises respectively an isomerthat is 2R, 3R; 2S, 3S or 2R, 3S.

Also provided by the present invention is a composition of matter havingthe structure:

wherein R₁ is H or comprises O, N, S, a phosphate group or a phosphonategroup, and any combination thereof; wherein R₂ is H, or comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbonchain, a branched carbon chain, a straight carbon chain comprising oneor more heteroatoms, a branched carbon chain comprising one or moreheteroatoms, a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₅is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₉ and R₁₀ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

Also provided by the present invention is a composition of matter havingthe structure:

wherein R₁ is H or comprises O, N, S, a phosphate group or a phosphonategroup, and any combination thereof; wherein R₂ is H, or comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbonchain, a branched carbon chain, a straight carbon chain comprising oneor more heteroatoms, a branched carbon chain comprising one or moreheteroatoms, a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₅is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₉and R₁₀ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

This invention also provides a method for monitoring the course ofpharmacokinetics of a drug after administration to a subject or patient,the process comprising administering to a subject or patient a drugcomprising any of the above described compounds, and detecting theadministered drug at selected intervals, thereby monitoring the courseof pharmacokinetics of the drug in the subject or patient.

These compounds are useful in a number of indications or diseaseconditions, including treatments for cancer, asthma, anaphylaxis,autophagy, central nervous system, including glioblastoma multiforme andothers. Additionally, this invention provides a method for treatingtumors of the central nervous system in which any of the above describedcompounds are administered to a subject or patient having a tumor ortumors of the central nervous system.

Other aspects of the compounds and methods of the present invention areprovided in further detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J. FIG. 1A shows decreased growth of U373 cells in serum-freemedium in the presence (filled squares) and in the absence (opencircles) of siRNA targeted to a specific sequence of SphK1 mRNA. FIG. 1Bshows decreased growth of U373 cells in medium containing serum in thepresence (filled squares) and in the absence (open circles) of siRNAtargeted to a specific sequence of SphK1 mRNA. FIG. 1C shows decreasedgrowth of LN229 cells in serum-free medium in the presence (filledsquares) and in the absence (open circles) of siRNA targeted to aspecific sequence of SphK1 mRNA. FIG. 1D shows decreased growth of LN229cells in medium containing serum in the presence (filled squares) and inthe absence (open circles) of siRNA targeted to a specific sequence ofSphK1 mRNA. FIG. 1E shows the drastic reduction of SphK1 in U373 andLN229 cells in the presence of siRNA targeted to a specific sequence ofSphK1 mRNA. FIG. 1F shows that expression of SphK1 in U373 cells is muchlower than expression of SphK1 in LN229 cells. FIG. 1G shows decreasedgrowth of U373 cells in serum-free medium in the presence of 3 μM(filled squares) and 10 μM SK1-I (filed diamonds) as compared to growthin the absence (open triangles) of SK1-I. FIG. 1H shows decreased growthof U373 cells in medium containing serum in the presence of 3 μM (filledsquares) and 10 μM SK1-I (filed diamonds) as compared to growth in theabsence (open triangles) of SK1-I. FIG. 1I shows decreased growth ofLN229 cells in serum-free medium in the presence of 3 μM (filledsquares) and 10 μM SK1-I (filed diamonds) as compared to growth in theabsence (open triangles) of SK1-I. FIG. 1J shows decreased growth ofLN229 cells in medium containing serum in the presence of 3 μM (filledsquares) and 10 μM SK1-I (filed diamonds) as compared to growth in theabsence (open triangles) of SK1-I.

FIGS. 2A-2D. FIG. 2A shows chemotaxis of LN229 cells toward serum, EGFand lysophosphatidic acid (LPA) is significantly inhibited by SK1-I.FIG. 2B shows that LPA, serum and EGF also stimulated in vitro invasionof LN229 cells into the basement membrane matrix Matrigel, which wasgreatly attenuated in the presence of SK1-I. FIG. 2C shows that SK1-Ireduced Akt phosphorylation at Thr308, Ser473, and p70S6Kphosphorylation at Thr 389 in LN229 cells stimulated by serum, LPA andEGF, but that SK1-I did not significantly affect EGF- or serum-inducedERK1/2 activation or serum-, LPA- or EGF-stimulated ERK1/2phosphorylation. FIG. 2D shows the reversal of inhibition of EGF-inducedAkt phosphorylation by SK1-I by addition of S1P, and that SK1-I did notaffect EGF-induced tyrosine phosphorylation of GEFR or Gab 1.

FIGS. 3A-3F. FIG. 3A shows a significant reduction in S1P levels within20 minutes after addition of SK1-I, and 70% reduction in S1P levelswithin 1 hour that was accompanied by increased sphingosine levels andno major changes in ceremide levels. FIG. 3B shows that increasedphosphorylation of JNK in LN229 cells after addition of SK1-I wasaccompanied by enhanced phosphorylation of c-JUN and ATF-2, and thatSP600125 blocked JNK activation as demonstrated by inhibition of c-Junand ATF-2 phosphorylation. FIG. 3C shows that 1 μM of SP600125 reversedSK1-I induced lethality of LN229 cells. FIG. 3D shows that addition of aspecific JNK peptide inhibitor significantly reversed the cytotoxiceffects of SK1-I, whereas a control peptide was ineffective. FIG. 3Eshows that addition of agents that perturb ERK1/2, Akt or INK signalinggreatly enhanced SK1-I lethality. FIG. 3F shows that expression ofdominant-negative MEK1 enhanced SK1-I induced LN229 cell death, whiledominant-negative Akt did not. In addition, expression ofconstitutively-activated AKT or MEK1 or expression of Bcl-xL suppressedcell death induced by SK1-I.

FIGS. 4A-4D. FIG. 4A shows that growth of GMB6 glioblastoma cells inserum-free medium is greatly reduced by SK1-I in a dose-dependentmanner. FIG. 4B shows that 10 μM SK1-I markedly reduced growth of GMB6cells in medium containing serum. FIG. 4C shows that SK1-I suppressedserum- and EGF-induced invasion of GBM6 cells. FIG. 4D shows that SK1-Ireduced basal and serum- and EGF-stimulated phosphorylation of Aktshortly following treatment without affecting pERK1/2 levels.

FIGS. 5A-5C. FIG. 5A shows that tumors resulting from subcutaneousinjection of LN229 cells into the flanks of mice that were subsequentlyeither treated with SK1-I or vehicle were significantly smaller in theSK1-I treatment group. FIG. 5B shows that treatment with SK1-I reducedtumor weight by almost 4-fold as compared to vehicle treated controls.FIG. 5C shows that tumor volume and size were reduced in mice treatedwith SK1-I as compared to vehicle treated controls.

FIG. 6 shows decreased vascularization, reductions in blood vesseldensity, greatly reduced VEGF expression, increased numbers of TUNELpositive apoptotic tumor cells, and markedly decreased levels ofphosphorylated Akt in tumors of mice treated with SK1-I as compared tovehicle treated controls.

FIGS. 7A-7D. FIG. 7A shows that mice implanted intracranially withGFP-labeled LN229 cells and subsequently treated with SK1-I showed notumors whereas vehicle treated mice showed a large tumor in the righthemisphere of the brain. FIG. 7B shows that gadolinium enhancementshowed a small tumor in the brain of one SK1-I mouse at the site ofinjection. FIG. 7C shows that at day 40 after intracranial implantationthe vehicle treated group began to show symptoms of tumor burden whilethe SK1-I treated group did not show any symptoms, and that Sk1-Iadministration showed significant survival benefit as compared tovehicle treated mice. FIG. 7D shows significantly fewer invading cellsand noticeable areas of necrosis in the middle of tumors in intracranialtumor sections from mice treated with Sk1-I compared to vehicle treatedanimals.

FIGS. 8A-8D. FIG. 8A shows distribution of F-actin across unstimulatedU373 cells as revealed by staining with Alexa488-conjugated phalloidin(left panels) and F-actin condensation at the leading edge withinlamellipodia in response to PMA exposure (right panels) in the absence(top panels) or in the presence (bottom panels) of siRNA targeted to aspecific sequence of SphK1 mRNA. FIG. 8B shows distribution of F-actinacross U373 cells as revealed by staining with Alexa488-conjugatedphalloidin (left panels), in cells in vehicle (middle panels) and inresponse to PMA exposure (right panels) in the absence (top panels) orin the presence (bottom panels) of SK1-I. FIG. 8C shows lamellipodialength of F-actin in unstimulated U373 cells and in U373 cells inresponse to PMA exposure in the absence or in the presence of siRNAtargeted to a specific sequence of SphK1 mRNA. FIG. 8D showslamellipodia length of F-actin in unstimulated U373 cells and in U373cells in response to PMA exposure in the absence or in the presence ofSK1-I.

FIGS. 9A-9B. FIG. 9A shows chemotaxis of U373 cells toward serum or EGFin Boyden chamber assays was reduced in the presence of SK1-I. FIG. 9Bshows that SK1-I reduced basal Akt phosphorylation, that serum and EGFenhanced phosphorylation of Akt in U373 cells and that Sk1-I did notreduce EGF- and serum-induced ERK1/2 activation in U373 cells.

FIGS. 10A-10D. FIG. 10A shows that treatment of LN229 cells with SK1-Iinduced apoptosis by increased cleavage of PARP. FIG. 10B shows thattreatment of LN229 cells with SK1-I increased fragmented and condensednuclei and delayed activation of JNK. FIG. 10C shows that treatment ofLN229 cells with SK1-I increased DNA strand breaks as detected by TUNELstaining. FIG. 10D shows that treatment of LN229 cells with SK1-Isuppressed long-term survival of the cells in clonogenic assays.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and analogs of the present invention are designed invarious forms, including their resemblance to the substrate, to theproduct formed by reaction of the substrate and enzyme, e.g.,sphingosine kinases including sphingosine kinase Type 1, and to anyintermediates formed in reaction. Reaction products are usuallycharacterized by low binding affinity, e.g., low Km. Still, by providingenough binding affinity to the reaction products, the compositions andanalogs of the present invention are useful and thereby produce usefulinhibitory or regulatory effects against the desired enzyme.

Preferential Inhibition of SphK1

The ability to identify compounds and analogs which preferentiallyinhibit or regulate SphK1 as opposed to SphK2 is desirable. Five foldand even ten fold greater inhibition of SphK1 over SphK2 is particularlyuseful.

The ability to inhibit SphK 1 differentially from SphK 2 allowsassessments of the individual SphK 1 and SphK 2 activities when bothactivities are present in a cell extract. This is easily carried out byan analysis of the amount of the total SphK activity (i.e.,transformation of Sph into Sph-P) in the absence of the inhibitor (whichshould be a composite of the individual SphK1 and SphK2 activities) andin the presence of the SphK 1 inhibitor where activity should only begenerated by the SphK 2. Since the total activity as well as thecontribution derived from SphK2 are known, a simple subtraction gives anestimate of the initial contribution by SphK1 in the assay carried outin the absence of the inhibitor. This is useful for diagnisitic orprognostic evaluations when viewed n the context of diseases where theselevels are abnormal compared to the healthy state of an individual.

The present invention provides a composition of matter having thestructure:

wherein R₁ is H or comprises N, S, a phosphate group or a phosphonategroup, and any combination thereof; wherein R₂ is H, or comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbonchain, a branched carbon chain, a straight carbon chain comprising oneor more heteroatoms, a branched carbon chain comprising one or moreheteroatoms, a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₄is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₅ comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing.

In the just described composition, one or more of the groups, R₂, R₃,R₄, R₅, R₆, R₇ or R₈, comprise at least one double bond or at least onetriple bond, or both at least one double bond and at least one triplebond.

In the just described composition, the straight carbon chain, thebranched carbon chain, the straight carbon chain comprising one or moreheteroatoms or the branched carbon chain comprising one or moreheteroatoms in R₂, R₃, R₄, R₅, R₆, R₇ or R₈ comprises an alkyl, asubstituted alkyl, an alkene, a substituted alkene, an alkyne or asubstituted alkyne, and combinations of any of the foregoing.

Also provided by this invention is a composition, as just described butwherein R₅ comprises at least one double bond, this composition havingthe structure:

wherein R₉ comprises H, a straight carbon chain, a branched carbonchain, a straight carbon chain comprising one or more heteroatoms, abranched carbon chain comprising one or more heteroatoms, a cyclic ring,a heterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing; and wherein R₁₀ comprises a straightcarbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing. Inthis composition, R₉ or R₁₀, or both R₉ and R₁₀ comprise at least onedouble bond or at least one triple bond, or at least one double bond andat least one triple bond.

Furthermore, in this composition just described, R₅ can comprise atleast one triple bond, such composition having the structure:

wherein R₉ comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing.

In the compositions above described, one or more of the groups, R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉ or R₁₀ comprise one or more halides.

Also provided by this invention is the composition in accordance withthose just described, wherein R₅ comprises an aromatic ring, thecomposition having the structure:

wherein R₁₁ comprises H, OH, a halide, a straight carbon chain, abranched carbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; an whereinR₁₁ and R₁₂ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

Additionally, in the composition just described, R₁₀ can comprise anaromatic group, the composition having the structure:

wherein R₁₁ comprises H, OH, a halide, a straight carbon chain, abranched carbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₁₁ and R₁₂ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

In the just described compositions, R₁₀, R₁₁, or both R₁₀ and R₁₁comprise one or more halides.

In another aspect of the above described compositions, R₃ and R₄independently can comprise the same or different R or S isomer. R₃ andR₄ can also together comprise an isomer that is 2R, 3R; 2S, 3S; 2R, 3S;or 2S, 3R.

Additionally, in the compositions above described, the C4 atom may beasymmetric and may comprise the 4R conformation or the 4S conformation.

In the compositions of the present invention, a detectable label can beincluded. Such a detectable label comprises a ligand or a fluorescentdye. The ligand can comprise but is not limited to biotin, digoxygeninor fluorescein. The fluorescent dye can assume a number of variouswell-known fluorescent dyes including fluorescein, fluoresceinisothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein,rhodamine, rhodamine 6G, rhodamine X, rhodol, sulforhodamine 101,tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate(TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC),dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo)benzoic acid (DABCYL), cyanine dyes or derivatives, and any combinationsof the foregoing.

Moreover, in the present compositions, the Cl atom can be asymmetric andcan comprise the 1R conformation. Alternatively, the Cl atom can beasymmetric and comprises the is conformation.

In the above compositions, the heteroatom or heteroatoms comprise S, Nor O, and combinations thereof. Such heteroatom or heteroatoms formlinkages which are well known in the art. Those skilled in the art willreadily appreciate such linkages which have been disclosed. See, forexample, U.S. Pat. No. 4,707,440. For illustration purposes only, thefollowing linkages are useful in accordance with this invention:

and a combination of any of the foregoing. Those skilled in the art willappreciate that such linkages can take the form just described, or theycan take a reverse or opposite form.

In the compositions above described, at least two of the groups, R₁, R₂,R₃, R₄ or R₅, can be joined together to form one or more rings. Thus,any of these groups can be cyclized to form additional rings betweensuch ring forming R groups. This cyclization through ring forming Rgroups can be carried out through conventional methods. Such joining ofthe individual R groups can be covalently or even non-covalent.

The invention herein also provides a composition of matter having thestructure:

wherein R₁ is H or comprises OH, N, S, a phosphate group or aphosphonate group, and any combination thereof; wherein R₂ comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing,wherein said R₂ is substituted with one or more halides; wherein R₃ is Hor comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₄is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₅ comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing.

In this just described composition, one or more of the groups, R₂, R₃,R₄, R₅, R₆, R₇ or R₈, can comprise at least one double bond or at leastone triple bond, or both at least one double bond and at least onetriple bond.

Furthermore, the straight carbon chain, the branched carbon chain, thestraight carbon chain comprising one or more heteroatoms or the branchedcarbon chain comprising one or more heteroatoms in R₂, R₃, R₄, R₅, R₆,R₇ or R₈ can comprise an alkyl, a substituted alkyl, an alkene, asubstituted alkene, an alkyne or a substituted alkyne, and combinationsof any of the foregoing.

In the just described composition, the group R₅ can comprise at leastone double bond, the composition having the structure:

wherein R₉ comprises H, a straight carbon chain, a branched carbonchain, a straight carbon chain comprising one or more heteroatoms, abranched carbon chain comprising one or more heteroatoms, a cyclic ring,a heterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing; and wherein R₁₀ comprises a straightcarbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing.

In the composition just described, the group R₉ or the group R₁₀, orboth R₉ and said R₁₀ can comprise at least one double bond or at leastone triple bond, or at least one double bond and at least one triplebond.

In the above composition wherein R₅ comprises at least one triple bond,the composition has the structure:

wherein R₉ comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing.

In the compositions above described, one or more of the groups, R₃, R₄,R₅, R₆, R₇, R₈, R₉ or R₁₀ comprises one or more halides.

In the above composition wherein R₅ comprises an aromatic ring, thecomposition has the structure:

wherein R₉ comprises H, a straight carbon chain, a branched carbonchain, a straight carbon chain comprising one or more heteroatoms, abranched carbon chain comprising one or more heteroatoms, a cyclic ring,a heterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing; and wherein R₁₁ comprises H, OH, a halide,a straight carbon chain, a branched carbon chain, a straight carbonchain comprising one or more heteroatoms, a branched carbon chaincomprising one or more heteroatoms, a cyclic ring, a heterocyclic ring,an aromatic ring or a hetero-aromatic ring, and any combination of theforegoing; and wherein R₁₁ and R₁₂ independently comprise a straightcarbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing.

In the above composition wherein R₁₀ comprises an aromatic group, thecomposition has the structure:

wherein R₁₁ comprises H, OH, a halide, a straight carbon chain, abranched carbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₁₁ and R₁₂ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

Furthermore, in the above described compositions wherein the group R₁₀,the group R₁₁, or both R₁₀ and R₁₁ comprise one or more halides.

Also, in accordance with this invention, the groups R₃ and R₄independently can comprise the same or different R or S isomer in theabove-described compositions. Moreover, the groups R₃ and R₄ cantogether comprise an isomer that is 2R, 3R; 2S, 3S; 2R, 3S; or 2S, 3R.

In the above described composition, the C4 atom can be asymmetric andcan comprise the 4R conformation. Alternatively, the C4 atom can beasymmetric and can comprises the 4S conformation.

The alkene containing compositions above can further comprise adetectable label. Such a detectable label can comprise a ligand or afluorescent dye. Where the former, the ligand can comprise biotin,digoxygenin or fluorescein. Where a fluorescent dye is contemplated asthe detectable label, the fluorescent dye can comprise fluorescein,fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM),naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodol,sulforhodamine 101, tetramethylrhodamine (TAMRA),tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine,eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin,methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL),cyanine dyes or derivatives, and any combinations of the foregoing. Theforegoing list of fluorescent dyes is illustrative and is not intendedto limit this invention.

Additionally, in these alkene compositions, the Cl atom may beasymmetric and may comprise the 1R conformation or the 1S conformation.

In these alkene compositions just described, the heteroatom orheteroatoms can comprise S, N or O, and combinations thereof. Asdescribed earlier, the heteroatom or heteroatoms can form a linkagecomprising any of the following linkages:

and a combination of any of the foregoing. Such linkages have beendescribed (see, e.g., U.S. Pat. No. 4,707,440) and are known to thoseskilled in the chemical arts. These linkages can take the form above, orthey can be used in a reverse or opposite orientation. Thus, the aboveform of such linkages is in no way intended to be limiting to thisinvention.

In the alkene containing composition, at least two of the groups, R₁,R₂, R₃, R₄ or R₅, can be joined together to form one or more ringsthrough cyclization as described above.

This invention additionally provides a composition of matter having thestructure:

wherein R₁ is H or comprises OH, N, S, a phosphate group or aphosphonate group, and any combination thereof; wherein R₂ is H, orcomprises a straight carbon chain, a branched carbon chain, a straightcarbon chain comprising one or more heteroatoms, a branched carbon chaincomprising one or more heteroatoms, a cyclic ring, a heterocyclic ring,an aromatic ring or a hetero-aromatic ring, and any combination of theforegoing; wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straightcarbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₆, R₇ and R₈ independently comprise H, a straight carbon chain,a branched carbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₄is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₅comprises a straight carbon chain, a branched carbon chain, a straightcarbon chain comprising one or more heteroatoms, a branched carbon chaincomprising one or more heteroatoms, a cyclic ring, a heterocyclic ring,an aromatic ring or a hetero-aromatic ring, and any combination of theforegoing; and wherein R₃ and R₄ each comprises respectively an isomerthat is 2R, 3R; 2S, 3S or 2R, 3S.

In another embodiment, one or more of said R₂, R₃, R₄, R₅, R₆, R₇ or R₈in the just described composition can comprise at least one double bondor at least one triple bond, or both at least one double bond and atleast one triple bond.

Furthermore, the straight carbon chain, the branched carbon chain, thestraight carbon chain comprising one or more heteroatoms or the branchedcarbon chain comprising one or more heteroatoms in R₂, R₃, R₄, R₅, R₆,R₇ or R₈ can comprise an alkyl, a substituted alkyl, an alkene, asubstituted alkene, an alkyne or a substituted alkyne, and combinationsof any of the foregoing.

Additionally, in this composition, the group R₅ can comprise at leastone double bond, so that the composition has the structure:

wherein R₉ comprises H, a straight carbon chain, a branched carbonchain, a straight carbon chain comprising one or more heteroatoms, abranched carbon chain comprising one or more heteroatoms, a cyclic ring,a heterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing; and wherein R₁₀ comprises a straightcarbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing. Inanother embodiment, the group R₉ or the group R₁₀, or both R₉ and R₁₀can comprise at least one double bond or at least one triple bond, or atleast one double bond and at least one triple bond.

Also contemplated by this invention is a composition as just describedbut where R₅ comprises at least one triple bond, such a compositionhaving the structure:

wherein R₉ comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring or a hetero-aromatic ring, and anycombination of the foregoing.

In the various just described compositions, one or more of the groupsR₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉ or R₁₀ can comprise one or more halides.

In a variation of this invention, the group R₅ can comprise an aromaticring so that the composition has the structure:

wherein R₁₁ comprises H, OH, a halide, a straight carbon chain, abranched carbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₁₁ and R₁₂ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

In yet a different variation, this invention provides the abovedescribed composition where the group R₁₀ comprises an aromatic group sothat the composition has the structure:

wherein R₁₁ comprises H, OH, a halide, a straight carbon chain, abranched carbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₁₁ and R₁₂ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

In the just described compositions, the groups R₁₀, R₁₁, or both R₁₀ andR₁₁ comprise one or more halides.

It should also be appreciated that in the above compositions, the C4atom may be asymmetric and may comprise the 4R conformation or the 4Sconformation.

As in other compositions of this invention, the last describedcompositions can further comprise a detectable label. Such detectablelabels are conventional and well known in the art. The detectable labelcan comprise a ligand or a fluorescent dye. In the case of the former,biotin, digoxygenin or fluorescein are contemplated and are useful. Iffluorescent dyes are contemplated, a number of such dyes can beemployed, including fluorescein, fluorescein isothiocyanate (FITC),6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine6G, rhodamine X, rhodol, sulforhodamine 101, tetramethylrhodamine(TAMRA), tetramethylrhodamineisothiocyanate (TRITC),4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl,hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoicacid (DABCYL), cyanine dyes or derivatives, and any combinations of theforegoing.

In another embodiment for the above described compositions, the Cl atommay be asymmetric and may comprise the 1R conformation or the 1Sconformation.

In these compositions just described, the heteroatom or heteroatoms cancomprise S, N or O, and combinations thereof. As earlier described, theheteroatom or heteroatoms can form a number of linkages including thoseof the form

and a combination of any of the foregoing.

As in the case of other compositions of the present invention, at leasttwo of the groups R₁, R₂, R₃, R₄ or R₅ can be joined together to formone or more rings. Cyclization and the formation of rings by joining Rgroups has been described above.

Also provided by the present invention is a composition of matter havingthe structure:

wherein R₁ is H or comprises O, N, S, a phosphate group or a phosphonategroup, and any combination thereof; wherein R₂ is H, or comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbonchain, a branched carbon chain, a straight carbon chain comprising oneor more heteroatoms, a branched carbon chain comprising one or moreheteroatoms, a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₅is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; and whereinR₉ and R₁₀ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

In another embodiment for the just described composition, one or more ofthe groups, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉ or R₁₀, can comprise at leastone double bond or at least one triple bond, or both at least one doublebond and at least one triple bond. In yet another embodiment, thestraight carbon chain, the branched carbon chain, the straight carbonchain comprising one or more heteroatoms or the branched carbon chaincomprising one or more heteroatoms in R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉ orR₁₀ can comprise an alkyl, a substituted alkyl, an alkene, a substitutedalkene, an alkyne or a substituted alkyne, and combinations of any ofthe foregoing. Moreover, one or more of the groups, R₂, R₃, R₄, R₅, R₆,R₇, R₈, R₉ or R₁₀, can comprise one or more halides.

It should also be appreciated that in these compositions, R₃ and R₄ canindependently comprise the same or different R or S isomer. Moreover, R₃and R₄ together can comprise an isomer that is 2R, 3R; 2S, 3S; 2R, 3S;or 2S, 3R. Furthermore, in these compositions, the C4 atom may beasymmetric and may comprise the 4R conformation or the 4S conformation.

These last described compositions can further comprise a detectablelabel, which are conventional and known in the art. Such detectablelabels can comprise a ligand or a fluorescent dye. In the case of theformer, biotin, digoxygenin or fluorescein are contemplated but shouldnot be considered limiting. In the case of the latter, many fluorescentdyes are known in the art, but for the sake of illustration, thefollowing are useful in accordance with this invention: fluorescein,fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM),naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodol,sulforhodamine 101, tetramethylrhodamine (TAMRA),tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine,eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin,methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL),cyanine dyes or derivatives, and any combinations of the foregoing.

In other aspects for these compositions, the Cl atom may be asymmetricand comprise the 1R conformation. Alternatively, the Cl atom may beasymmetric and comprise the is conformation.

In the last described compositions of the present invention, theheteroatom or heteroatoms can comprise S, N or O, and combinationsthereof. Furthermore, such heteroatom or heteroatoms can form a numberof linkages, including the following list which should be consideredlimiting but only illustrative:

and a combination of any of the foregoing.

In these compositions described above, at least two of the groups, R₁,R₂, R₃, R₄ or R₅, can be joined together to form one or more ringsthrough the cyclization of R groups using conventional methods ofchemical synthesis.

This invention also provides a composition of matter having thestructure:

wherein R₁ is H or comprises O, N, S, a phosphate group or a phosphonategroup, and any combination thereof; wherein R₂ is H, or comprises astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring or a hetero-aromatic ring, and any combination of the foregoing;wherein R₃ is H or comprises OH, NR₆R₇, N⁺R₆R₇R₈, a straight carbonchain, a branched carbon chain, a straight carbon chain comprising oneor more heteroatoms, a branched carbon chain comprising one or moreheteroatoms, a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₆,R₇ and R₈ independently comprise H, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₅is H or comprises OH, a halide, ═O, a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing; wherein R₉and R₁₀ independently comprise a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring or ahetero-aromatic ring, and any combination of the foregoing.

In the last described composition, one or more of the groups, R₂, R₃,R₄, R₅, R₆, R₇, R₈, R₉ or R₁₀, can comprise at least one double bond orat least one triple bond, or both at least one double bond and at leastone triple bond. Furthermore, the straight carbon chain, the branchedcarbon chain, the straight carbon chain comprising one or moreheteroatoms or the branched carbon chain comprising one or moreheteroatoms in R₂, R₃, R₄, R₅, R₆, R₇ or R₈ can comprise an alkyl, asubstituted alkyl, an alkene, a substituted alkene, an alkyne or asubstituted alkyne, and combinations of any of the foregoing. Moreover,one or more of the groups, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉ or R₁₀, cancomprise one or more halides.

In other embodiments for this composition, R₃ and R₄ can independentlycomprise the same or different R or S isomer. In a further aspect, R₃and R₄ can together comprise an isomer that is 2R, 3R; 2S, 3S; 2R, 3S;or 2S, 3R.

As described for other compositions of the present invention, these lastcompositions can also comprise a detectable label which are conventionaland known in the art. Such detectable labels can comprise a ligand or afluorescent dye. In the case of the former, biotin, digoxygenin orfluorescein are contemplated but are not intended to be limiting. In thecase of the latter, many fluorescent dyes are known. For purposes ofillustration, these can include fluorescein, fluorescein isothiocyanate(FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine,rhodamine 6G, rhodamine X, rhodol, sulforhodamine 101,tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate(TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC),dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo)benzoic acid (DABCYL), cyanine dyes or derivatives, and any combinationsof the foregoing.

The Cl atom in these compositions may be asymmetric and may comprise the1R conformation or the 1S conformation.

In further aspects, the heteroatom or heteroatoms can comprise S, N orO, and combinations thereof. Such heteroatom or heteroatoms can form alinkage which are described in the art. For illustration purposes only,these linkages can comprise any of the following

and a combination of any of the foregoing. Moreover, these linkages cantake the above configuration, or they can be used in reverse or oppositeconfigurations. In other words, the orientation can be varied and is notlimited to the precise form shown above.

As described earlier, in these last compositions, at least two of thegroups, R₁, R₂, R₃, R₄ or R₅, can be joined together to form one or morerings. Such cyclization and ring formation using R groups in thecompounds and analogs can be carried out using conventional methods. Therings can be joined covalently or non-covalently.

Pharmacokinetics and Patient Management

Another important aspect of the present invention is a method formonitoring the course of pharmacokinetics of a drug after administrationto a subject or patient. In this method, a drug comprising any of thecompounds described above is administered to a subject or patient. Theadministered drug can be detected at selected intervals, therebymonitoring the course of pharmacokinetics of the drug. Such a method isuseful in studying and monitoring patient management because the courseand progress of the administered drug can be followed within cells,tissues, organs or the subject or patient as a whole. By attachingsignaling moieties to the compounds and analogs of this invention, invivo imaging can be carried out following the administration of the drugto detect the presence of the drug within cells of the subject orpatient. Cell staining can also be carried out to locate the presence ofthe drug within cells of the subject or patient's sample or specimen.Radioactivity in the form of radioactively labeled drugs, i.e.,compounds or analogs, can also be utilized.

Indications/Diseases

Yet another important feature of this invention is a method for treatingtumors of the central nervous system. This method comprises the step ofadministering to a subject or patient having a tumor or tumors of thecentral nervous system any of the above-described compounds of thepresent invention. The tumors of the central nervous system can comprisevarious forms known in the art, including glioblastoma, and moreparticularly, glioblastoma multiforme (GBM). Useful in the treatment ofGBM is the compound below which comprises

The above-described compounds and analogs of the present invention areuseful for treatment and monitoring of a number of indications anddiseases. In Ser. No. 12/387,228, filed Apr. 29, 2009 (contentsincorporated by reference), the following indications and diseases aredisclosed: killing or damaging cancer cells (leukemia cells, breastcancer cells, prostate cancer cells, pancreatic cancer cells, gliomacancer cells, colon cancer cells, lung cancer cells, ovarian cancercells, melanoma cells, renal cancer cells); causing cancer cells toundergo apoptosis; inhibiting growth, metastasis and development ofchemoresistance in cancer cells, treating or reducing symptoms ofleukemia; increasing the ability of anticancer agent to kill cancercells; inhibiting survival signaling in cancerous cells; attenuatingimmune reactivity; and reducing symptoms of multiple sclerosis.

In addition to the above-named indications and diseases, the compoundsand analogs of the present invention are useful for diseases associatedwith neural cell death or muscular cell death, such as Parkinson'sdisease, Alzheimer's disease, amniotropic lateral sclerosis and musculardystrophy, AIDS, fulminant hepatitis, and diseases linked todegeneration of the brain (e.g., Creutzfeld-Jakob disease, retinitispigmentosa and cerebellar degeneration, myelodysplasis (e.g., aplasticanemia, ischemic diseases such as myocardial infarction and stroke,hepatic diseases such as alcoholic hepatitis, hepatitis B and hepatitisC, joint diseases such as osteoarthritis, atherosclerosis, alopecia,damage to the skin due to UV light, lichen planus, atrophy of the skin,cataract and graft rejections. These compounds and analogs describedherein are applicable to immunopathology caused by influenza virus.Other diseases include those implicating caveolar endocytosis, plasmamembrane microdomain formation, transmembrane signaling or integrinfunction (e.g., inflammatory diseases including cancer, MS,prothrombotic risk, ulcerative colitis and renal disease). These mightoccur as the result of infection by certain bacteria, fungi or viralspecies, e.g., SV40 virus.

Among other uses of the compounds and analogs of the present inventionare inhibition of angiogenesis in tumors, modulation of the immunesystem by altering lymphocyte trafficking for treatment of autoimmunediseases or prolongation of allograft transplant survival, andpreventing, inhibiting or treating neuropathic pain. Also within thescope of use for the present compounds and analogs are the treatment orprevention of disorders or syndromes including cell proliferativedisorders, e.g., cancer, ischemia or restenosis. The compounds andanalogs of the present invention can also be used to screen for amodulator of disorders/syndromes including the aforementioned cellproliferative disorders (cancers, ischemia or restenosis).

These compounds and analogs are applicable to treating or attenuatingcomplications in subjects or patients suffering from trauma or sepsis.

Other pathological conditions can be addressed through the use andapplication of the present compounds and analogs, includingcardiovascular diseases, diabetes, stroke, autoimmune and inflammatorydiseases, allergic diseases such as dermatitis, T helper-1, relateddiseases, chronic obstructive pulmonary disease, asthma, cancer andneurodegenerative disorders, some of which have already been describedabove.

The following examples are offered by way of illustration and not by wayof limitation to the present invention.

Example 1: Synthesis of BML-258

The compound described and used below, BML-258, was synthesizedaccording to the following protocol and procedures.

BML-258 Synthetic Protocol

To 4-n-pentylphenylacetylene 1 (3.343 g, 0.01776 mol) in 65 mL dry THFat −20° C. under an atmosphere of N2 was added n-BuLi (10.2 mL of 1.6Min hexanes, 0.01628 mol) dropwise. The reaction mixture was stirred at−20° C. for 2 hours. Methyl(R)-(+)-3-(t-butoxycarbonyl)-2,2-dimethyl-4-oxazolidinecarboxylate 2(3.393 g, 0.01480 mol) in 25 mL dry THF was added via cannula/N2. Thereaction was stirred overnight at −20° C. overnight. TLC (20% ethylacetate/hexanes) indicated completeness of reaction. The mixture wasdiluted with Et₂O and carefully washed with water and brine. Flashcolumn chromatography (12% Ethyl acetate/hexanes, silica gel) yielded4.50 g (73%) of a mixture of erythro and threo products. PreparativeHPLC (Dynamax Si, 15% Ethyl acetate/hexanes, 260 nm) yielded 3.71 gerythro 3 and 0.49 g threo. 1H NMR (CDCl₃) erythro: 7.34-7.32 (d, 2H),7.12-7.09 (d, 2H), 5.19-5.16 (d, 1H), 4.73-4.70 (d, 1H), 4.26-3.96 (m,3H), 2.61-2.56 (t, 2H), 1.62 (s, 3H), 1.60-1.50 (m, 2H), 1.54 (s, 3H),1.50 (s, 9H), 1.34-1.27 (m, 4H), 0.91-0.86 (t, 3H).

To oxazolidine 3 (3.48 g, 0.00814 mol) in 100 mL MeOH was addedAmberlyst-15 (200 mg). The reaction was stirred overnight at roomtemperature. TLC (30% Ethyl acetate/hexanes) indicated completeness ofreaction. The mixture was filtered and flash chromatographed (5%MeOH/methylene chloride, silica gel) to give 2.44 g (79%) ofaminoalcohol 4. 1H NMR (CDCl₃): 7.34-7.32 (d, 2H), 7.12-7.09 (d, 2H),5.45-5.38 (d, 1H), 4.88-4.82 (m, 1H), 4.25-4.19 (m, 1H), 3.91- 3.80 (m,2H), 3.26-3.23 (d, 1H), 2.61-2.56 (t, 2H), 1.63-1.54 (m, 2H), 1.49 (s,9H), 1.35-1.26 (m, 4H), 0.91-0.86 (t, 3H).

To alkyne 4 (2.44 g, 0.00646 mol) in 125 mL dry Et₂O at 0° C. under anatmosphere of N2 was added Red-Al (9.85 mL of 65 wt % in toluene,0.03232 mol) dropwise. The reaction was allowed to warm to roomtemperature following the addition and was stirred for 36 hours. TLC(40% Ethyl acetate/hexanes) indicated completeness of reaction. Thereaction was cooled to 0° C. and carefully quenched with 15% NaOHsolution. This mixture was stirred vigorously until both layers wereclear (45 min). The layers were separated and the aqueous layerextracted with chloroform (3×). The combined organic layers were washedwith 15% NaOH, water and brine. Flash chromatography (gradient of 5%MeOH/methylene chloride to 20% MeOH/methylene chloride+1% NH₄OH, silicagel) yielded 1.76 g (72%) of trans alkene 5. 1H NMR (CDCl₃): 7.31-7.29(d, 2H), 7.15-7.12 (d, 2H), 6.70-6.65 (d, 1H, J=16 Hz), 6.26-6.18 (dd,1H, J=16 Hz), 5.35- 5.32 (d, 1H), 4.55-4.49 (m, 1H), 4.03-3.96 (m, 1H),3.80-3.68 (m, 2H), 2.83-2.79 (d, 1H), 2.61-2.56 (t, 2H), 1.65-1.55 (m,2H), 1.44 (s, 9H), 1.34-1.25 (m, 4H), 0.91-0.86 (t, 3H).

To BOC-alkene 5 (0.350 g, 0.00092 mol) in 20 mL dry THF under anatmosphere of N₂ was carefully added DIBAL (9.22 mL of 1M in THF,0.00922 mol) at room temperature. Following the addition, the reactionwas brought to reflux. After 24 hours of reflux, the mixture was cooledto room temperature and an additional 5.0 mL DIBAL solution (0.00500mol) was added. Reflux was resumed for another 24 hours. The reactionwas cooled to 0° C. and carefully quenched with water (0.60 mL), 15%NaOH (0.60 mL) and water again (1.50 mL). THF (50 mL) was added and themixture stirred vigorously for 15 minutes. Na₂SO₄ (2 g) and celite (2 g)were then added and stirring was continued for 30 minutes while warmingto room temperature. The mixture was filtered and the filter cakeextracted with copious THF. Flash chromatography (gradient of 2%MeOH/methylene chloride to 10% MeOH/methylene chloride+0.75% NH₄OH)yielded 0.187 g (73%) of amine 6. 1H NMR (CDCl₃): 7.31-7.29 (d, 2H),7.15-7.12 (d, 2H), 6.68-6.63 (d, 1H, J=16 Hz), 6.22-6.14 (dd, 1H, J=16Hz), 4.51-4.47 (m, 1H), 3.80-3.74 (m, 3H), 2.61-2.56 (t, 2H), 2.50 (s,3H), 2.40-2.10 (broad, 2H), 1.65-1.55 (m, 2H), 1.34-1.25 (m, 4H),0.91-0.86 (t, 3H). HRMS (MH+): Calc. −278.2120, Found −278.2119.

To amine 6 (0.335 g, 0.00121 mol) in 15 mL dry Et₂O at 0° C. was added3.0 mL of 1M HCl/Et₂O. A white precipitate formed immediately. Afterstirring for 15 minutes at room temperature, the precipitate wasfiltered and washed with Et₂O to give 0.325 g (89%) of BML-258. 1H NMR(DMSO): 8.75-8.50 (bd, 2H), 7.38-7.34 (d, 2H), 7.19-7.15 (d, 2H),6.65-6.60 (d, 1H, J=16 Hz), 6.30-6.22 (dd, 1H, J=16 Hz), 5.84-5.82 (m,1H), 5.30-5.25 (m, 1H), 4.60-4.54 (m, 1H), 3.76-3.72 (m, 2H), 3.18-3.10(m, 1H), 2.64 (s, 3H), 2.56-2.50 (t, 2H), 1.60-1.50 (m, 2H), 1.34-1.23(m, 4H), 0.90-0.85 (t, 3H).

SK1-I,(2R,3S,4E)-N-methyl-5-(4′-pentylphenyl)-2-aminopent-4-ene-1,3-diol(BML-258), was synthesized by BIOMOL International (Plymouth Meeting,Pa.) as described in Example 1. Sphingosine and N,N-dimethylsphingosinewere obtained from BIOMOL. [γ-³²P]ATP (3000 Ci/mmol) was purchased fromPerkin Elmer (Boston, Mass.). Boc-D-FMK (BOC), Z-VAD-FMK (ZVAD) andetoposide were from EMD Biosciences (San Diego, Calif.). Terminaldeoxynucleotidyl transferase Br-dUTP nick end labeling (TUNEL) kit forflow cytometry was from Sigma Aldrich (St. Louis, Mo.). TUNEL kit forimmunohistochemistry was from Roche Applied Science (Indianapolis,Ind.). FITC-4 labeled annexin V/propidium iodide staining kit forapoptosis was from BD Biosciences (San Jose, Calif.).

Example 2: General Procedure for Synthesis of SK1-I Analogs from Alkynes

To alkyne, 1, (1.2 eq) in dry THF at −20° C. was added n-BuLi (1.1 eq.of 1.6M hexanes) drop wise. The reaction was stirred at −20° C. for 2hours. The aldehyde, 2, (1 eq., dissolved in dry THF, was added dropwise. The reaction was placed in a −20° C. freezer overnight. Afterapproximately 18 hours, the reaction was diluted with diethyl ether andwashed with water and brine. Flash column chromatography yielded amixture of erythro and threo products. The pure erythro compound, 3, wasisolated via HPLC.

The resulting erythro oxazolidine, 3, was stirred with Amberlyst 15resin in methanol overnight to remove the acetonide protecting group.Flash column chromatography yielded the Boc protected aminodiol, 4.

To alkyne aminodiol, 4, (1 eq.) in diethyl ether at 0° C. was addedRed-Al (5 eq. of 65 wt % in toluene) drop wise. The reaction was allowedto warm to room temperature overnight. After approximately 18 hours ofreaction, the mixture was cooled to 0° C. and quenched with 15% NaOH (5eq). Flash column chromatography yielded the alkenyl aminodiol 5.

To alkenylaminodiol, 5, (1 eq) in dry THF at 0° C. was added DIBAL (10eq. of 1M/THF) drop wise. Following the addition, the reaction wasgradually warmed to room temperature and then refluxed overnight. After24 hours of reaction, the mixture was cooled to 0° C. and quenchedsuccessively with water (4 eq), 15% NaOH (4 eq) and water (10 eq). Flashcolumn chromatography yielded the target compound, 6.

Analogs of general structure, 7, are prepared using the appropriatelysubstituted alkyne following the general procedure. R=3,4-dimethoxy;4-phenyl; 3-pentyl.

Synthesis of SK1-I Analogs with Various N-Alkyl Groups

The appropriately substituted BOC-protected SK1-I analog is synthesizedusing the general procedure outlined previously.

To BOC-protected SKI-1, 8, (1 eq) in methanol at 0° C. is bubbledhydrogen chloride gas until the mixture is saturated. The reaction isstirred at room temperature until TLC indicates completeness ofreaction. The resulting solution is evaporated to dryness and dissolvedin dry pyridine. Acetic anhydride (1 eq.) is added and the reactionstirred at room temperature until complete by TLC. Flash chromatographyyielded the monoacetyl derivative, 9.

To N-acetyl-SKI-1, 9, (1 eq) in dry THF at 0° C. is added LithiumAluminum Hydride (4 eq. of 1M/THF). Following the addition, the reactionmixture is warmed to room temperature and then refluxed overnight. Themixture was then cooled to 0° C. and quenched successively with water (4eq), 15% NaOH (4 eq) and water (10 eq). Flash column chromatographyyielded the target alkylamine, 10.

Synthesis of Di-N-Alkyl SK1-I Analogs

To SK1-I, 11, (1 eq) in dry THF at room temperature was added methyliodide (1 eq). The reaction was stirred until TLC indicated completereaction. Flash column chromatography yielded the desired compound, 12.

Materials and Methods

Cell culture. U373-MG and LN229 human glioblastoma cells (ATTC,Manassas, Va.) were cultured in DMEM supplemented with 5% FCS. Primaryhuman non-established glioblastoma GBM6 cells were kindly provided byDr. C. David James and were passaged as tumors in nude mice andsubcultured for 1 week following isolation from tumors in mediacontaining 2% FCS to prevent growth of contaminating rodent fibroblastsand then cultured in 5% FCS as described (Yacoub et al., Mol Cancer Ther7:314-329, 2008). LN229 cells were transfected with H2B-EGFP plasmid andstable colonies were isolated following selection with 1 mg/ml of G418.LN229-H2B-EGFP cells were passaged as tumors as described above.

Xenograft tumors. Adult male NCI nu/nu mice were purchased from NCI(Frederick, Md.). All animal studies were conducted in the AnimalResearch Core Facility at VCU School of Medicine in accordance with theinstitutional guidelines. LN229 cells (1×10⁶) were injected in theflanks (4 sites per mouse). Palpable tumors appeared in about one week.Five days later, when tumors reached 3-4 mm in diameter, mice wererandomly separated into 2 groups and injected i.p. with saline or SK1-I(10 mg/kg) every other day. Tumor measurements were made with calipers,and tumor volume was calculated using the formula: (π×[length inmillimeters]×[width in millimeters]2)/6. At the end of the experiment,the animals were euthanized and the tumors removed, fixed in formalinand embedded in paraffin, or frozen in liquid nitrogen.

Intracranial LN229 xenograft tumors. Adult female NCI nu/nu mice wereanesthetized and LN229-H2B-EGFP cells (2.5×10⁴ in 1 μl PBS) werestereotactically implanted in the putamen region (1 mm anterior and 2.5mm lateral to the Bregma at the depth of 3.5 mm at a rate of 0.1μl/min). Mice were monitored for recovery until complete wakening. 20days after implantation, mice were injected i.p. with SK1-I (20 mg/kg inPBS) every other day. Mice were observed daily following tumorimplantation and were euthanized on reaching a moribund state.

Details about infection of cells with recombinant adenoviruses, cellproliferation and cell death assays, immunohistochemistry,immunocytochemistry, and confocal microscopy are presented in theinformation below.

Results

SK1-I Potently Inhibits Growth and Survival of Human Glioblastoma Cells

Previous studies demonstrated that S1P and SphK1, the kinase thatproduces it, play critical roles in growth and survival of glioblastomacells (Van Brocklyn et al., J Neuropathol Exp Neurol 64:695-705, 2005;Van Brocklyn et al., Cancer Lett 181:195-204, 2002; and Radeff-Huang etal. J Biol Chem 282:863-870, 2007). In agreement, downregulation ofSphK1 expression decreased growth of both U373 cells, which expressmutated PTEN, and LN229 cells expressing wild type PTEN, in serum-freemedium (FIGS. 1A, 1C) as well as in the presence of serum (FIGS. 1B,1D), which greatly enhanced their growth. Expression of SphK1 in thesecells was drastically reduced by siRNA targeted to a specific sequenceof SphK1 mRNA, as detected by western blotting with a polyclonalanti-SphK1 antibody (FIG. 1E). The greater sensitivity of U373 cells todownregulation of SphK1 might be due to much lower SphK1 expression andenzymatic activity compared to LN229 cells (FIG. 1F).

The first SphK1-specific inhibitor, SK1-I, was recently described (Paughet al., Blood 283:3365-3375, 2008). SK1-I inhibited growth of both U373(FIGS. 1G, 1H) and LN229 (FIGS. 1I, 1J) cells in a dose-dependentmanner. A significant inhibitory effect was observed at 3 μM. SK1-I at10 μM strongly inhibited growth of U373 and LN229 cells cultured in theabsence of serum (FIGS. 1G, 1I). SK1-I was less effective when cellswere cultured in the presence of serum, which contains multiple growthfactors and S1P. However, even in the presence of serum, within 2-4days, there were severe reductions in cell numbers after treatment with10 μM SK1-I (FIGS. 1H, 1J).

SK1-I Inhibits Migration and Invasion of Glioblastoma Cells

As S1P and SphK1 have been shown to regulate migration and invasion ofglioblastoma cells (Lepley et al. Cancer Res 65:3788-3795, 2005; Younget al. Exp Cell Res 313:1615-1627, 2007; Van Brocklyn et al. Cancer Lett199:53-60, 2003; and Malchinkhuu et al. Oncogene 24:6676-6688, 2005),and SphK1 regulates actin cytoskeletal dynamics (Kusner et al. J BiolChem 282:23147-23162, 2007) and lamellipodia formation (Maceyka et al.Mol Cell Biol 28:5687-5697, 2008), it was of interest to examine whetherinhibition of SphK1 by SK1-I correlated with changes in reorganizationof the actin cytoskeleton. F-actin was distributed across unstimulatedU373 cells, as revealed by staining with Alexa488-conjugated phalloidin(FIG. 8A). In response to PMA the actin cytoskeleton underwent robustreorganization, and more F-actin was condensed at the leading edgewithin structures termed lamellipodia (FIG. 8A). In agreement with aprevious study with human macrophages (Kusner et al., J Biol Chem282:23147-23162, 2007), downregulation of SphK1 markedly reduced thenumber of actin-rich lamellipodia produced by treatment with PMA (FIGS.8A, 8C). Similarly, inhibiting SphK1 with SK1-I dramatically reducedPMA-stimulated F-actin reorganization at the leading edge and formationof lamellipodia and induced disassembly of filopodia (FIGS. 8B, 8D).

These results support the notion that SphK1 activity is required foractin filament dynamics (Kusner et al. J Biol Chem 282:23147-23162,2007). Therefore, the effect of SK1-I on migration and invasion ofglioma cells was next examined. Directed motility (chemotaxis) of U373cells toward serum or EGF in Boyden chamber assays was reduced by SK1-1(FIG. 9A). Similarly, chemotaxis of LN229 cells, which show much greaterrates of basal and stimulated migration toward serum and EGF than U373cells, is also significantly inhibited by SK1-I (FIG. 2A). SK1-I alsodrastically inhibited chemotaxis of LN229 cells toward lysophosphatidicacid (LPA), another serum-borne lysophospholipid that has been shown tobe a potent chemoattractant for certain glioblastoma cell lines,including LN229 cells (Malchinkhuu et al. Oncogene 24:6676-6688, 2005)(FIG. 2A). LPA, serum, and EGF also stimulated in vitro invasion ofLN229 cells (FIG. 2B), determined by their ability to invade thebasement membrane matrix Matrigel, which was also greatly attenuated bySK1-I (FIG. 2B).

SK1-I Reduces Basal and Stimulated Akt Phosphorylation

S1P-induced glioblastoma cell proliferation is greatly suppressed byinhibition of ERK1/2 and PI3K/Akt pathways (Van Brocklyn et al. CancerLett 181:195-204, 2002). Thus, it was of interest to examine the effectsof SK1-I on these signaling pathways. We utilized phospho-specificantibodies to examine phosphorylation of Akt at Thr308 in the activationloop and at Ser473 at the C-terminus, which are required for fullactivation (Haas-Kogan et al. Curr. Biol 8:1195-1198, 1998). Consistentwith their expression of wild-type PTEN, LN229 cells have low basal Aktphosphorylation, which was rapidly increased by serum, LPA, and EGF, toa lesser extent (FIG. 2C). SK1-I reduced Akt activation induced by allthree stimuli. Treatment with SK1-I for only 20 minutes markedlysuppressed phosphorylation of Akt at both Thr308 and Ser473 (FIG. 2C).SK1-I also reduced activation of p70S6K (Thr389), a downstream target ofAkt. In sharp contrast, although serum, LPA, and EGF stimulated ERK1/2,in these short-term assays, SK1-I did not significantly affectstimulated ERK1/2 phosphorylation at Thr202/Tyr204 (FIG. 2C). Moreover,although Akt is active in U373 cells because, like many human gliomasthey express a nonfunctional mutant form of PTEN that does not inhibitthe PI3K/Akt pathway (Haas-Kogan et al. Curr Biol 8:1195-1198, 1998),SK1-I reduced their basal Akt phosphorylation at Thr308 and Ser473 (FIG.9B). A significant inhibitory effect was observed within 20 min (FIG.9B), which lasted for at least 24 hours (data not shown). As expected,serum and EGF enhanced phosphorylation of Akt, whereas SK1-I reduced it(FIG. 9B). The inhibitory effect of SK1-I on Akt phosphorylation was notdue to its degradation as there were no significant reductions in totalAkt levels after treatment with SK1-I. However, SK1-I did not reduceEGF- and serum-induced ERK1/2 activation in both U373 (9B) and LN229cells (FIG. 2C).

To substantiate that the effects of SK1-I were due to its ability toinhibit SphK1, S1P add-back experiments were carried out. Consistentwith the reduction in levels of S1P by SK1-I (FIG. 3A), inhibition ofEGF-induced Akt phosphorylation by SK1-I was reversed by addition of SIP(FIG. 2D). EGF has been shown to activate PI3K/Akt by phosphorylatinggrowth factor receptor-bound protein 2 (Grb2)-associated binder 1(Gab 1) (Mattoon et al. BMC Biol 2:24-35, 2004). However, SK1-I did notaffect EGF-induced tyrosine phosphorylation of EGFR or of Gab1 (FIG.2D), indicating that SK1-I did not directly interfere with EGFRactivation. Thus, the SphK1 inhibitor SK1-I specifically inhibitsphosphorylation and activation of Akt in GBM cells in a S1P-dependentmanner.

Because downregulation of SphK1 not only decreases S1P, it alsoincreases ceramide levels (Maceyka et al. J Biol Chem 280:37118-37129,2005; Pchejetski et al. Cancer Res 65:11667-11675, 2005; Taha et al.FASEB J 20:482-484, 2006; Berdyshev et al. Cell Signal 18:1779-1792,2006), it was of interest to examine the effects of inhibition of SphK1with SK1-I on these sphingolipid metabolites that have been reported tohave opposing effects on cell growth and apoptosis (Cuvillier et al.Nature 381:800-803, 1996; and Hannun et al. Nat Rev Mol Cell Biol9:139-150, 2008). There was a significant reduction in S1P levels within20 min after addition of SK1-I (FIG. 3A), which correlated with therapid inhibition of Akt phosphorylation. Furthermore, within 1 h afteraddition of SK1-I, S1P levels were dramatically decreased by 70% thatwas accompanied by an increase in sphingosine levels without majorchanges in ceramide levels (FIG. 3A). However, after 24 h of treatmentwith SK1-I, ceramide levels increased markedly, particularlypro-apoptotic C16-ceramide. Unlike safingol (L-threo-dihydrosphingosine)(Coward et al. Autophagy 5:184-193, 2009), a pan SphK inhibitor, onlyless than 1% of SK1-I was converted to the tri-N-methyl metabolite after24 h (FIG. 3A) and no other metabolites were detected. Moreover, incontrast to its structural analogue, the immunosuppressant drug FTY720,SK1-I is not readily phosphorylated, ruling out potential actionsthrough S1P receptors.

Inhibition of c-Jun N-Terminal Kinase Attenuates SK1-I-Induced CellDeath

In agreement with many previous studies showing that downregulation ofSphK1 and ceramide elevation are associated with increased apoptosis(reviewed in (Hannun et al. Nat Rev Mol Cell Biol 9:139-150, 2008;Cuvillier, O. Expert Opin Ther Targets 12:1009-1020, 2008; and Shida etal. Curr Drug Targets 9:662-673, 2008), treatment with SK1-I inducedapoptosis of LN229 cells as demonstrated by increased cleavage of PARP(FIG. 10A), a substrate for caspase-mediated proteolysis duringapoptosis, increased fragmented and condensed nuclei (FIG. 10B), andincreased DNA strand breaks detected by TUNEL staining (FIG. 10C).Moreover, SK1-I markedly suppressed long-term survival of LN229 cells inclonogenic assays (FIG. 10D).

Sphingolipid metabolites, S1P versus sphingosine and ceramide, usuallyhave opposing effects on Akt and the stress-related c-Jun NH2-terminalkinase (JNK) pathways (Cuvillier et al. Nature 381:800-803, 1996; andHannun et al. Nat Rev Mol Cell Biol 9:139-150, 2008). Concomitant withthe inactivation of the cytoprotective Akt pathway, exposure of LN229cells to SK1-I was accompanied by delayed activation of JNK (FIG. 10B),without affecting p38 MAPK (data not shown). Increased phosphorylationof JNK after addition of SK1-I was accompanied by enhancedphosphorylation of its substrates, the transcription factors c-Jun(Ser63/73) and ATF-2 (Thr71) (FIG. 3B).

The output of ERK1/2 and Akt signaling versus JNK signaling represents akey homeostatic mechanism that in many cells regulates the balancebetween cell survival and cell death processes (Xia et al. Science270:1326-1231, 1995). Thus, the effects of a variety of agents thatperturb these signaling pathways on SK1-I mediated lethality was nextexamined. Inhibition of MEK1/2, PI3K, and p38 by U0126, LY294002,SB202190, respectively, enhanced SK1-I lethality, whereas inhibition ofJNK by SP600125 markedly attenuated the effects of SK1-I in both U373and LN229 cells (FIG. 3E and data not shown). As expected, SP600125efficiently blocked JNK activation, as demonstrated by inhibition ofc-Jun and ATF-2 phosphorylation (FIG. 3B). Even at 1 μM, a concentrationbelieved to specifically inhibit JNK without having non-specific effectson other kinases, SP600125 markedly reversed SK1-I-induced lethality(FIG. 3C). The importance of the INK pathway using a specific INKpeptide inhibitor was further examined. The INK peptide inhibitor blocksthe activation domain of INK and prevents phosphorylation of c-Jun. Thispeptide also significantly reversed the cytotoxic effects of SK1-I (FIG.3D), whereas the control peptide was ineffective. Similarly, expressionof dominant-negative MEK1 also enhanced SK1-I-induced LN229 cell death,while dominant-negative Akt did not (FIG. 3F). Moreover, expression ofconstitutively-activated Akt or MEK1, or expression of Bcl-xL,suppressed cell death induced by SK1-I (FIG. 3F).

Effect of SK1-I on Primary Non-Established Glioblastoma

Observations with SK1-I to primary non-established human GBM6glioblastoma cells was expanded. GBM6 glioblastoma cells have been shownto produce invasive, diffuse tumors in the brains of mice (Giannini etal. Neuro-oncol 7:164-176, 2005; and Yacoub et al. Cancer Biol Ther7:917-933, 2008). GBM6 express mutant p53, wild-type PTEN, and EGFRvIII,a constitutively activated mutant form of EGFR (Yacoub et al. CancerBiol Ther 7:917-933, 2008; and Yacoub et al. Cancer Biol Ther 3:739-751,2004). Similar to LN229 and U373 cells, growth of GBM6 cells was greatlyreduced by SK1-I (FIG. 4A). In the absence of serum there was adose-dependent effect of SK1-I and significant growth inhibition wasobserved at a concentration as low as 1 μM (FIG. 4A). Moreover, as withthe glioblastoma cell lines, 10 μM SK1-I markedly reduced growth of GBM6in the presence of serum (FIG. 4B). SK1-I also suppressed serum- andEGF-induced invasion of GBM6 (FIG. 4C). Similar to the effects on theestablished glioblastoma cell lines, SK1-I reduced basal and serum- andEGF-stimulated phosphorylation of Akt shortly following treatment,without affecting pERK1/2 levels (FIG. 4D).

SK1-I Reduces Tumor Growth in Mice

Encouraged by these findings, the effect was examined of SK1-I onsubcutaneous tumor growth of LN229 cells, which are fairly invasive andgrow phenotypically similar to invasive gliomas in situ (Nakamizo et al.Cancer Res 65:3307-3318, 2005). Tumors appeared as palpable masses aboutone week after subcutaneous injection of one million cells in the flankof a mouse (FIG. 5A). Five days later, when the tumor size could bereliably measured (3-4 mm in diameter), animals were randomized andSK1-I was injected intraperitoneally every other day at a dose of 10mg/kg. Tumors in control animals showed significant increases in volumeas early as day 27, and growth accelerated thereafter. Statisticalanalysis (single factor ANOVA) revealed significantly smaller tumors inthe SK1-I treatment group (FIG. 5A). After 43 days, animals had to besacrificed due to the tumor burden in control mice. Tumors were excised,weighed, and histology examined. In addition to the tumor volume andsize (FIGS. 5A, 5C), SK1-I treatment reduced tumor weight by almost4-fold (FIG. 5B) and decreased vascularization in tumors, as shown byhematoxilin and eosin staining (FIG. 6 ). Similar reductions in bloodvessel density were observed by staining with antibodies to themouse-specific endothelial cell marker CD31 (FIG. 6 ). In agreement,immunohistochemistry for VEGF also revealed elevated expression of thisangiogenic factor in vehicle treated tumors that was greatly reduced inSK1-I treated mice (FIG. 6 ). The disruption of tumor cyto-architectureby SK1-I was accompanied by increased numbers of TUNEL positiveapoptotic tumor cells (FIG. 6 ). In agreement with attenuation of Aktphosphorylation in GBM cells by SK1-I, immunostaining for phosphorylatedAkt in tumors was markedly decreased by treatment with SK1-I (FIG. 6 ).

SK1-I Enhances Survival of Mice with LN229 Orthotopic Tumors

It was of interest to also examine whether SK1-I was effective in themore clinically relevant orthotopic model of intracranially implantedLN229 cells. On the basis of trial growth rate analyses, intraperitonealtreatment with SK1-I was initiated at day 20 after intracranialimplantation of GFP-labeled LN229 cells when the tumors would beestablished and the mice would be expected to be asymptomatic. Animalsin the vehicle treated group began to show symptoms of tumor burden atday 40 and were euthanized on reaching a moribund state between day 43and 49 (FIG. 7C). None of the SK1-I treated mice showed any symptoms atthis point and SK1-I administrations was then halted (FIG. 7C). At day48, T2W MRI revealed the presence of a large tumor in the righthemisphere of vehicle treated mice (FIG. 7A), while no tumors wereevident in SK1-I treated mice (FIG. 7A). Gadolinium enhancement revealeda small tumor in the brain of this SK1-I treated mouse at the site ofthe injection (FIG. 7B). Moreover, visualization of GFP-labeled LN229cells in intracranial tumor sections showed significantly fewer invadingcells and noticeable areas of necrosis in the middle of the tumors fromSK1-I treated animals compared to vehicle treated animals (FIG. 7D).Kaplan-Meier survival analysis of intracranial glioblastoma xenograftsshowed significant survival benefit from SK1-I administration comparedto vehicle control animals (FIG. 7C), indicating that SK1-I wasremarkably efficacious in the brain even when administeredintraperitoneally.

Discussion

Currently available therapies only minimally improve the prognosis ofGBM patients and new therapeutic targets are desperately needed.Accumulating evidence suggests that SphK1 is an attractive new target.SphK1 message and protein levels are upregulated in GBM (Van Brocklyn etal. J Neuropathol Exp Neurol 64:695-705, 2005) and in astrocytomatissues compared to adjacent normal brain (Li et al. Clin Cancer Res14:6996-7003, 2008). Patients whose tumors were among the highestone-third with regard to SphK1 expression survived a median of 102 days,whereas those within the lower two-thirds survived a median of 357 days(Van Brocklyn et al. J Neuropathol Exp Neurol 64:695-705, 2005). Highexpression of SphK1 was shown to be a predictor of poor prognosis forastrocytoma patients (Li et al. Clin Cancer Res 14:6996-7003, 2008).

Here targeting SphK1 with SK1-I has been shown to suppress proliferationof several human glioblastoma cell lines, including U373, LN229, U87,and U118 cells as well as non-established GBM6 cells. SK1-I alsopotently induced apoptosis and inhibited invasion of these cells.Similar to the effects of SK1-I, downregulation of SphK1 expression hasbeen shown to reduce glioblastoma cell growth, survival, migration, andinvasion (Van Brocklyn et al. J Neuropathol Exp Neurol 64:695-705,2005). SK1-I was effective in GBM that are mutant for PTEN or p53 orhave a constitutively activated form of EGFR. This is particularlyimportant since more than 80% of GBMs show strong Akt activation, manydue to lost or mutated PTEN. Activation of EGFR is also a criticalpathogenetic event, with amplifications, mutations, or rearrangementscommonly observed (Wen et al. N Eng J Med 359:492-507, 2008). SK1-I alsoshowed significant antitumor activity in vivo, inducing GBM tumor cellapoptosis and reducing tumor vascularization.

The mechanisms by which inhibition of SphK1 by SK1-I so profoundlyreduces proliferation and survival of GBM in vitro and inhibits tumorgrowth in vivo is now beginning to become unraveled. SK1-I rapidlysuppresses phosphorylation of Akt and its targets p70S6K and GSK3□, andthus interferes with signaling through the Akt pathway, which isfrequently activated in gliomagenesis (Wen et al. N Eng J Med359:492-507, 2008). This inhibition by SK1-I is not due to a directeffect on Akt, as it did not inhibit Akt activity in an in vitro kinaseassay (Paugh et al. Blood 112:1382-1391, 2008). It is also well acceptedthat S1P produced by activation of SphK1 is released from cells andstimulates its receptors that are linked to activation of Akt. Indeed,the reduction of S1P levels by SK1-I is rapid and could contribute todecreased phosphorylation of Akt. The effects of SK1-I may not bemediated solely by reduction of “inside-out signaling” by S1P but alsoby reduction of intracellular S1P. These results are consistent withprevious reports showing that SphK1 and intracellular S1P are criticalfor Akt activation and cell proliferation independently of S1P receptors(Radeff-Huang et al. J Biol Chem 282:863-870, 2007; and Oliviera et al.J. Biol Chem 278:46452-46460, 2003). Moreover, in 1321N1 glioblastomacells, DNA synthesis and cyclin D expression was increased in a SphK1-and Akt-dependent manner independently of S1P receptors (Radeff-Huang etal. J Biol Chem 282:863-870, 2007). In agreement, overexpression ofSphK1 promotes cell survival and growth even in cells devoid offunctional S1PRs (Olivier et al., ibid.). Similarly, overexpression ofSphK1 is a S1P receptor-independent oncogenic event in progression oferythroleukemia that involves activation of Akt (Le Scolan et al. Blood106:1808-1816, 2005). In agreement with previous results in leukemiacells (Paugh et al. Blood 112:1382-1391, 2008), SK1-I not only inhibitedS1P production in glioma cells, it also increased levels of itspro-apoptotic precursor ceramide that has been shown to cause growthinhibition and apoptosis by inhibiting Akt (Hannun et al. Nat Rev MolCell Biol 9:139-150, 2008). Thus, biphasic inhibition of Akt is likelydue to a rapid decrease in intracellular S1P and later sustainedincreases in ceramide. Furthermore, a recent study in glioma cellsshowed that inhibition of the Akt pathway strongly upregulated ceramidelevels by inhibiting conversion of ceramide to complex sphingolipids dueto reduction of ER to Golgi trafficking of ceramide (Giussani et al. JBiol Chem 284:5088-5096, 2009). Because ceramide in turn furtherinhibits Akt, this engages a vicious cycle that amplifies the apoptoticeffect of SK1-I. Activation of JNK may also be due to inhibition of Aktfollowing SK1-I treatment as several studies raised the intriguingpossibility that the ability of Akt to inhibit JNK signaling is due tophosphorylation of specific targets in this pathway (Kim et al. Mol CellBiol 21:893-901, 2001; and Barthwal et al. J Biol Chem 278:3897-3902,2003).

Downregulation of SphK1, similar to SK1-I, causes a marked elevation inlevels of ceramide (Maceyka et al. J Biol Chem 280:37118-37129, 2005;Pchejetski et al. Cancer Res 65:11667-11675, 2005; Taha et al. FASEB J20:482-484, 2006; Berdyshev et al. Cell Signal 18:1779-1792, 2006).Consistent with the higher expression of SphK1 in GBM, ceramide levelsare lower in human gliomas compared to surrounding brain tissue, and areinversely related to tumor progression and short patient survival(Riboni et al. Glia 39:105-113, 2002). Thus, actions of SphK1 might berelated to its role in regulation of ceramide levels.

The existence of redundant survival pathways suggests that targeting asingle dysregulated pathway may not be sufficient to eliminate tumors.Indeed, it has been suggested that effective GBM therapy may requirecombinations of inhibitors targeting multiple signaling pathways(Stommel et al. Science 318:287-290, 2007). The finding of the presentinvention that inhibiting SphK1 with SK1-I further enhanced glioblastomacell lethality induced by inhibitors of other important signalingpathways that are frequently dysregulated in GBM may have implicationsfor the design of protocols combining SphK1 inhibitors together withconventional anticancer agents or experimental therapeutics.

Supplementary Materials and Methods

Reagents. S1P was obtained from BIOMOL (Plymouth Meeting, Pa.). Serumand medium were from Biofluids (Rockville, Md.). EGF was from LifeTechnologies (Gaithersburg, Md.). Anti-phospho Akt (Ser473 and Thr308),anti-Akt, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-ATF2(Thr71), anti-phospho-JNK (Thr183/Tyr185) antibodies were from CellSignaling (Beverly, Mass.) and anti-ERK2, anti-phospho-c-Jun (Ser63/73)and anti-clathrin heavy chain (CHC) antibodies were from Santa Cruz(Santa Cruz, Calif.). Rabbit polyclonal SphK1 antibodies were describedpreviously (Hait et al. J Biol Chem 280:29462-29469, 2005). Horseradishperoxidase (HRP)-conjugated and fluorescently labeled secondaryantibodies were from Jackson ImmunoResearch (West Grove, Pa.) andMolecular Probes (Eugene, Oreg.), respectively. Control andSphK1-specific siRNAs (Hait et al., ibid.) were obtained from Qiagen(Valencia, Calif.). WST-1 cell proliferation reagent and TUNEL kit forimmunohistochemistry were from Roche Applied Science (Indianapolis,Ind.). SK1-I((2R,3S,4E)-N-methyl-5-(4′-pentylphenyl)-2-aminopent-4-ene-1,3-diol,BML-258), was synthesized as the HCl salt by BIOMOL International (nowEnzo Life Sciences International, Plymouth Meeting Pa.). SP600125 andSB202190 were from Sigma-Aldrich (St. Louis, Mo.), JNK peptide inhibitor1 and the negative control peptide were from EMD Biosciences (San Diego,Calif.), and LY294002 and U0126 were from Cell Signaling Technology.

Downregulation of SphK1. U373-MG and LN229 cells were transfected with100 nM control siRNA or siRNA against SphK1 (sequence targeted:GGGCAAGGCCTTGCAGCTC [SEQ ID NO:1]), using Dharmafect 1 reagent(Dharmacon, Chicago, Ill.) as described (Paugh et al. FASEB J22:455-465, 2008).

SphK1 activity. SphK1 activity was determined exactly as described (Haitet al. J Biol Chem 280:29462-29469, 2005).

Western blotting. Cells were scraped in buffer containing 20 mMTris-HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 30 mM Na₄P₂O₇, 20 mM2-glycerophosphate, 1 mM Na₃VO₄, 5 mM EDTA, 2 mM EGTA, 0.5% SDS andprotease inhibitor cocktail (1:200 dilution) and probe-sonicated. Equalamounts of protein determined with bicinchoninic acid (Pierce, Rockford,Ill.), were resolved by SDS-PAGE and transferred to nitrocellulosemembranes. Blots were blocked with 5% non-fat dry milk in Tris-bufferedsaline containing 0.1% Triton X-100 (TBST) for 1 h at room temperature,and then incubated with primary antibodies (1:3,000 in 1% BSA)overnight, followed by the appropriate HRP-conjugated secondaryantibodies (1:40,000 in 1% BSA). Immunocomplexes were visualized byenhanced chemiluminescence (Pierce).

Immunohistochemistry. Paraffin sections were dewaxed, rehydrated,incubated with proteinase K before permeabilization, and then stainedwith hematoxylin-eosin. Frozen sections were dried, fixed in formalin,and stained with antibodies against mouse CD-31 (BD Pharmingen, SanJose, Calif.), or rabbit antibodies against phospho-Akt (Ser473) (CellSignaling) followed by immunohistochemistry with Alexa-488-conjugatedspecies-specific secondary antibodies. Cryosections were air dried,permeabilized with 0.5% Triton X-100, and stained with a fluoresceinTUNEL labeling kit (Roche Applied Sciences, Indianapolis, Ind.) followedby counterstaining with Hoechst. Sections were also stained with goatanti-human VEGF affinity-purified antibody (R&D), visualized withanti-goat horseradish peroxidase-diaminobenzidene staining kit (R&D) andcounterstained with hematoxylin.

Immunocytochemistry and confocal microscopy. Cells were grown on3-aminopropyl-triethoxysilane-treated 15 mm glass cover slips in 24 wellplates. Following treatments, cells were washed with PBS, fixed with 3%paraformaldehyde for 10 min at room temperature, and blocked in TBSTbuffer containing 1% BSA. After washing, cells were incubated in thesame buffer containing Alexa-conjugated phalloidin for 30 min, followedby 15 min incubation in 10 μg/ml of Hoechst 33342. For TUNEL assays,fixed cells were permeabilized with 0.5% TX-100 for 30 min, washed andincubated in TdT buffer supplemented with 250 μM CoCl₂, 20 units TdT(NEB, Ipswich, Mass.) and 1 nM fluorescein-12-dUTP (Roche) for 1 h at37° C. Coverslips were washed with TBST, rinsed in water, air dried andmounted on glass slides with Cytoseal 60 polymer (Richard-AllanScientific, Kalamazoo, Mich.). Images were collected on an LSM 510 laserconfocal microscope (Zeiss, Thornwood, N.Y.) with a 100× oil immersionobjective.

Cell proliferation and cell death assays. Cells were plated at 10,000cells/well in 48-well plates and allowed to attach for 24 h. Cellproliferation was measured at the indicated times with WST-1 andabsorbance was measured in a plate reader at 450 nm with backgroundsubtraction at 630 nm. Cell death was detected by trypan blue exclusionassays in which the percent of blue dye incorporating cells weredetermined using a light microscope and a hemacytometer as described(Yacoub et al. Mol Cancer Ther 7:314-329, 2008). Apoptotic cell deathwas measured by staining cell nuclei with the Hoechst dye bisbenzimideand apoptotic cells were identified by condensed, fragmented nuclearregions as described previously (Sankala et al. Cancer Res67:10466-10474, 2007). A minimum of 300 cells was scored.

Colony formation assay. Cells were plated at a density of 1000cells/well in a 12-well plate in DMEM containing 5% serum. After 8 h,SK1-I was added and 2 h later, the media was changed. After 10 days,cells were fixed in 4% paraformaldehyde and stained with crystal violet(0.05%). Colonies larger then 0.5 mm in diameter were counted.

Infection of cells with recombinant adenoviruses. Cells were plated at3×10³ per cm² and infected after 24 h (at a multiplicity of infection of50) with a control empty vector virus (CMV) or adenoviruses expressingconstitutively active (ca) Akt, dominant-negative (dn) Akt, caMEK1,dnMEK1, or Bcl-xL (Vector Biolabs, Philadelphia, Pa.).

Invasion and chemotaxis assays. Boyden chamber invasion assays werecarried out essentially as described (Shida et al. Cancer Res68:6569-6577, 2008).

Mass spectrometric analyses. Lipids were extracted and phosphorylatedand unphosphorylated sphingoid bases, individual ceramide acyl chainspecies, as well as SK1-I and its metabolites were quantified by liquidchromatography, electrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS) as described previously (Merrill et al. Methods36:207-224, 2005).

Statistical analysis. Experiments were repeated at least three timeswith consistent results. For each experiment, data from triplicatesamples were expressed as means±S.D. Statistics were performed by singlefactor ANOVA, and p<0.05 was considered significant. The Kaplan-Meierestimator was used to generate the survival curves and to estimate themedian survival values. Differences between survival curves werecompared using a log-rank test.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application, arehereby incorporated by reference in their entireties.

Many obvious variations will no doubt be suggested to those of ordinaryskill in the art, in light of the above detailed description andexamples of the present invention. It will be appreciated by thoseskilled in the art that any arrangement which is calculated to achievethe same purpose may be substituted for the specific embodiments shown.This application and invention are intended to cover any adaptations orvariations of the present invention. All such variations are fullyembraced by the scope and spirit of the invention as more particularlydefined in the claims that now follow.

What is claimed is:
 1. A method for treating neuropathic pain in apatient, comprising: administering to a patient in need of treatment forneuropathic pain a compound having the formula


2. The method of claim 1, wherein the administering step comprisesadministering

to the patient.
 3. The method of claim 2, wherein the step ofadministering

to the patient comprises administering

HCl to the patient.
 4. A method for treating neuropathic pain in apatient, comprising: administering to a patient in need of treatment forneuropathic pain a compound having the formula

wherein R comprises a straight carbon chain, a branched carbon chain, astraight carbon chain comprising one or more heteroatoms, a branchedcarbon chain comprising one or more heteroatoms, a cyclic ring, aheterocyclic ring, an aromatic ring, a hetero-aromatic ring, or anycombination of the foregoing.
 5. The method of claim 4, wherein R is astraight carbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, a branched carbon chain comprisingone or more heteroatoms, a cyclic ring, a heterocyclic ring, an aromaticring, a hetero-aromatic ring, or any combination of the foregoing. 6.The method of claim 5, wherein R is a straight carbon chain, a branchedcarbon chain, a straight carbon chain comprising one or moreheteroatoms, a branched carbon chain comprising one or more heteroatoms,a cyclic ring, a heterocyclic ring, an aromatic ring, or ahetero-aromatic ring.
 7. The method of claim 6, wherein R is a straightcarbon chain, a branched carbon chain, a straight carbon chaincomprising one or more heteroatoms, or a branched carbon chaincomprising one or more heteroatoms.
 8. The method of claim 7, wherein Ris a straight carbon chain or a branched carbon chain.
 9. The method ofclaim 8, wherein R is a straight carbon chain.
 10. A method for treatingneuropathic pain in a patient, consisting essentially of: administering

to the patient.